A Library of Tunable, Portable, and Inducer-Free Promoters Derived from Cyanobacteria

Cyanobacteria are emerging as hosts for various biotechnological applications. The ability to engineer these photosynthetic prokaryotes greatly depends on the availability of well-characterized promoters. Inducer-free promoters of a range of activities may be desirable for the eventual large-scale, outdoor cultivations. Further, several native promoters of cyanobacteria are repressed by high carbon dioxide or light, and it would be of interest to alter this property. We started with PrbcL and PcpcB, the well-characterized native promoters of the model cyanobacterium Synechococcus elongatus PCC 7942, found upstream of the two abundantly expressed genes, Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase, and phycocyanin β-1 subunit, respectively. The library of 48 promoters created via error-prone PCR of these 300-bp-long native promoters showed 2 orders of magnitude dynamic range with activities that were both lower and higher than those of the wild-type promoters. A few mutants of the PrbcL showed greater strength than PcpcB, which is widely considered a superstrong promoter. A number of mutant promoters did not show repression by high CO2 or light, typically found for PrbcL and PcpcB, respectively. Further, the wild-type and mutant promoters showed comparable activities in the fast-growing and stress-tolerant strains S. elongatus PCC 11801 and PCC 11802, suggesting that the library can be used in different cyanobacteria. Interestingly, the majority of the promoters showed strong expression in E. coli, thus adding to the repertoire of inducer-free promoters for this heterotrophic workhorse. Our results have implications in the metabolic engineering of cyanobacteria and E. coli.蓝细菌（Cyanobacteria）正逐渐成为各类生物技术应用的宿主菌株。对这类光合原核生物进行基因工程改造的能力，很大程度上依赖于经过充分表征的启动子的可用性。具备不同活性强度的无诱导剂启动子，或许是实现大规模户外培养的理想选择。此外，蓝细菌的多款内源启动子会受到高二氧化碳或强光的抑制，因此改造这一特性具有重要研究价值。本研究以模式蓝细菌长聚球藻PCC 7942（Synechococcus elongatus PCC 7942）中两款经过充分表征的内源启动子PrbcL与PcpcB为起点，这两个启动子分别位于两个高表达基因——核酮糖-1,5-二磷酸羧化酶/加氧酶（Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase）与藻蓝蛋白β-1亚基——的上游区域。通过对这两段长度为300 bp的内源启动子进行易错PCR（error-prone PCR）构建得到的包含48个启动子的突变文库，展现出两个数量级的动态活性范围，其活性涵盖了低于与高于野生型启动子的区间。部分PrbcL突变体的活性强度甚至高于被广泛视为超强启动子的PcpcB。多款突变启动子不再受高二氧化碳或强光的抑制——而这两种抑制效应分别是野生型PrbcL与PcpcB的典型特征。此外，在快速生长且耐胁迫的长聚球藻菌株PCC 11801与PCC 11802中，野生型与突变型启动子的活性表现相近，这表明该文库可应用于不同的蓝细菌菌株。值得注意的是，大多数启动子在大肠杆菌（E. coli）中均表现出较强的表达活性，从而扩充了这类异养模式工程菌可用的无诱导剂启动子工具库。本研究结果可为蓝细菌与大肠杆菌的代谢工程改造提供重要参考依据。